Myelodysplastic syndrome with transformation to acute monocytic leukemia with FLT3-ITD mutation following orthotopic liver transplantation

A rare case of a 46-year-old man who underwent myelodysplastic syndrome, acute monocytic leukemia with FLT3-ITD mutation and splenic disruption following orthotopic liver transplantation is reported. The study of this case may be helpful to understand both the pathogenesis of acute leukemia and new complication of liver transplantation.     

DNA载体介导RNA干扰抑制LMP-1基因对鼻咽癌细胞转移能力的影响

目的探讨应用DNA载体介导RNA干扰技术,抑制鼻咽癌细胞中EB病毒潜伏膜蛋白1（LMP-1）表达的可能性,观察LMP-1基因沉默对鼻咽癌细胞转移能力的影响。方法将能转录出干扰RNA的DNA模板插入pAVU6＋27质粒,构建能够介导RNA干扰的发夹状干扰RNA（shRNA）表达载体。采用磷酸钙共沉淀法,将质粒导入EB病毒（＋）的鼻咽癌细胞C611,筛选LMP-1基因沉默的克隆,通过观察其贴壁生长能力、基底膜穿透能力和移动能力的变化,分析LMP-1基因沉默对鼻咽癌细胞转移能力的影响。结果在shRNA表达载体有效转染的C611细胞中,LMP-1基因的表达受到稳定抑制。肿瘤细胞转移实验表明,LMP-1基因沉默的C611细胞,贴壁生长能力提高,基底膜穿透能力和细胞移动能力均明显下降。结论shRNA表达载体能够在鼻咽癌细胞内介导RNA干扰,并获得对LMP-1基因稳定的抑制。LMP-1基因可能通过贴壁生长能力、基底膜穿透能力和细胞移动能力等,影响鼻咽癌细胞的转移。     

Growth inhibition of carcinogen-transformed MCF-12F breast epithelial cells and hormone-sensitive BT-474 breast cancer cells by 1alpha-hydroxyvitamin D5

Several studies have established the active form of vitamin D(3) as an effective tumor-suppressing agent; however, its antitumor activity is achieved at doses that are hypercalcemic in vivo. Therefore, less calcemic vitamin D(3) analog, 1alpha-hydroxy-24-ethyl-cholecalciferol (1alpha[OH]D5), was evaluated for its potential use in breast cancer chemoprevention. Previously, 1alpha(OH)D5 showed anticarcinogenic activity in several in vivo and in vitro models. However, its effects on growth of normal tissue were not known. The present study was conducted to determine the effects of 1alpha(OH)D5 on the growth of normal mouse mammary gland and normal-like human breast epithelial MCF-12F cells and to compare these effects with carcinogen-transformed MCF-12F and breast cancer cells. No significant difference was observed in the growth or morphology of cultured mouse mammary gland and MCF-12F cells in the presence of 1alpha(OH)D5. However, the transformed MCF-12F cells underwent growth inhibition (40-60%, P < 0.05) upon 1alpha(OH)D5 treatment as determined by cell viability assays. Cell cycle analysis showed marked increase (50%) in G-1 phase for cells treated with 1alpha(OH)D5 compared with the controls. Moreover, the percentage of cells in the synthesis (S) phase of cell cycle was decreased by 70% in transformed MCF-12F, BT-474 and MCF-7 cells. The growth arrest was preceded by an increase in expression of cell cycle regulatory proteins p21(Waf-1) and p27(Kip-1). In addition, differential expression studies of parent and transformed MCF-12F cell lines using microarrays showed that prohibitin mRNA was increased 4-fold in the transformed cells. These results indicate that the growth inhibitory effect of 1alpha(OH)D5 was achieved in both carcinogen-transformed MCF-12F and breast cancer cells at a dose that was non-inhibitory in normal-like breast epithelial cells.     

Reversal of p53 epigenetic silencing in multiple myeloma permits apoptosis by a p53 activator

Inactivation of the p53 pathway is a common feature of neoplasia. Dysregulation of the p53 pathway has been shown to involve mutations of p53, increased expression of the p53 inhibitor HDM-2, or epigenetic silencing of the p53 promoter. In multiple myeloma, a neoplasia of terminally differentiated B cells, p53 mutations and deletions are relatively rare and occur in late stage disease. Here, we show that the p53 promoter is hypermethylated in several multiple myeloma cell lines in comparison to normal plasma cells. Two cell lines containing mutant p53, Lp-1 and OPM-2, show a methylation pattern that suggests that they contain one methylated and one unmethylated mutant allele. Two other cell lines, KMS-11 and OPM-2, show hypermethylation of p53 with a lack of expression. In all cell lines tested, treatment with a demethylating agents results in higher expression of p53. Furthermore, following increased expression of p53, treatment of the myeloma cell lines with a p53 activating peptide induces apoptosis. Therefore, combinatorial treatment with demethylating agents followed by delivery of a p53 activating peptide may be an effective therapeutic strategy against multiple myeloma.     

Autoantibodies to Ca2+ binding protein Calnuc is a potential marker in colon cancer detection

Calnuc is a calcium (Ca2+) binding protein found in both Golgi and cytoplasm, and it may play a role in G protein- and Ca2+-regulated signal transduction events. This study was designed to investigate the possibility of whether Calnuc protein might be a tumor-associated antigen (TAA) that induces autoantibody response in human cancers, and to evaluate the feasibility of the Calnuc antigen-antibody system as a marker in cancer detection. Purified full-length recombinant Calnuc protein was used as an antigen in enzyme-linked immunoassay and Western blotting for the detection of autoantibodies in cancers. Sera from 447 patients with 9 different types of cancer were analyzed. Although the frequency of autoantibody to Calnuc was found to be 4.7% in total groups of cancer, it was not significantly different to that of normal individuals (1.2%). However, the frequency of autoantibody to Calnuc in colon cancer (11.5%) was significantly higher than that in normal individuals (1.2%). The expression analysis of Calnuc in multiple colon cancer tissues by immunohistochemistry on tissue array further confirmed the high specificity of Calnuc in colon cancer. Of 69 colon cancer tissue specimens examined, 41 tissues (59.4%) overexpressed Calnuc, while normal colon tissues did not show any expression of Calnuc. The subcellular distribution analysis of Calnuc examined by subcellular fractionation and immunofluorescence indicates that Calnuc is a membrane associated protein and mostly distributed in Golgi, which is consistent with previous reports. With adding Calnuc into a TAA array (including p53, c-myc, cyclin B1, cyclin D1), the cumulative frequency of antibody to multiple TAAs in colon cancer was raised to 65.4% which is significantly higher than the cumulative frequency in normal individuals (6.1%). This indicates that a mini-array of multiple TAAs which includes Calnuc might provide a novel non-invasive approach to enhance antibody detection for colon cancer diagnosis.     

DNA damage response: the players, the network and the role in tumor suppression

One of the most common features of cancer is genetic instability. In response to numerous DNA-damaging insults, normal cells have evolved a complex mechanism to monitor and repair DNA damage lesions to maintain genomic integrity. The defects in DNA damage response, indeed, have been shown to associate closely with tumorigenesis. This review provides an overview on the molecular events in DNA damage signaling pathway, including cell cycle checkpoint and DNA repair. The recent research discoveries on how dysfunction in DNA damage response contributes to genomic instability and cancer development are also discussed.     

Targeting mutant (V600E) B-Raf in melanoma interrupts immunoediting of leukocyte functions and melanoma extravasation

Polymorphonuclear neutrophils (PMN) facilitate melanoma cell extravasation under dynamic flow conditions by the binding of intercellular adhesion molecule-1 (ICAM-1) on melanoma cells to beta2 integrins on PMNs, which is mediated by endogenously produced chemokine interleukin 8 (IL-8) from the tumor microenvironment. However, little is known about the role of B-Raf, the most mutated gene in malignant melanomas, in this process. In this study, we investigated the functional importance of B-Raf in melanoma extravasation by using short interfering RNA to reduce expression/activity of mutant (V600E)B-Raf in melanoma. Results indicated that knockdown of mutant (V600E)B-Raf inhibited melanoma cell extravasation in vitro and subsequent lung metastasis development in vivo. Mechanistic studies showed that inhibition of (V600E)B-Raf significantly reduced the constitutive secretion of IL-8 from melanoma cells as well as the capacity of endogenous IL-8 production from the melanoma-PMN microenvironment. Furthermore, a reduction in ICAM-1 expression on melanoma cells was detected following mutant (V600E)B-Raf knockdown. Together, these results suggest that targeting mutant (V600E)B-Raf reduces melanoma cell extravasation by decreasing IL-8 production and interrupting ICAM-1-beta2 integrin binding of melanoma cells to the endothelium mediated by PMNs in the microcirculation, which provides a rationale and mechanistic basis for targeting mutant (V600E)B-Raf to inhibit melanoma extravasation and subsequent metastasis development.     

白细胞介素12转染树突状细胞后与新建卵巢上皮性癌细胞融合的免疫原性研究

目的研究白细胞介素（IL）12转染入树突状细胞（DC）后,与新建卵巢上皮性癌细胞融合形成融合细胞,分析融合细胞的体外免疫杀伤效应。方法以新鲜卵巢上皮性癌组织建立卵巢上皮性癌细胞系,并进行鉴定。体外诱导产生人脐血DC。将IL-12与pcDNA3．1（＋）质粒连接后,分别通过脂质体法和电穿孔法将IL-12-pcDNA3．1（＋）质粒转染DC,再使用聚乙二酸法将新建卵巢上皮性癌细胞与转染IL-12的DC融合,用四甲基偶氮唑蓝法检测融合细胞的体外免疫杀伤效应。结果（1）形态学观察和免疫组化法检测结果均证实,新建卵巢上皮性癌细胞系为原代卵巢上皮性癌细胞。（2）人脐血培养7-10d,贴壁细胞出现大量毛刺状突起;免疫荧光染色显示,脐血DC主要组织相容性复合物Ⅱ类分子人白细胞抗原（HLA）-DR的阳性表达率为99％,共刺激分子B7-2的阳性表达率为50％。（3）RT—PCR技术检测结果证实IL—12转染DC成功。将转染IL—12的DC与新建卵巢上皮性癌细胞融合,RT—PCR技术和免疫荧光染色分析结果均证实融合细胞形成。（4）将转染IL—12的DC和新建卵巢上皮性癌的融合细胞、DC和新建卵巢上皮性癌的融合细胞分别与患者的外周血单个核细胞共同培养,激活T淋巴细胞,结果显示,两种活化后的T淋巴细胞均能杀伤新建卵巢上皮性癌细胞,且前者作用更强。结论转染IL—12后的DC与新建卵巢上皮性癌细胞融合后形成的融合细胞能激活T淋巴细胞,且其体外免疫杀伤效应更强。     

Nanofibrous poly(acrylonitrile-co-maleic acid) membranes functionalized with gelatin and chitosan for lipase immobilization

Nanofibrous membranes with an average diameter of 100 and 180 nm were fabricated from poly(acrylonitrile-co-maleic acid) (PANCMA) by the electrospinning process. These nanofibrous membranes contain reactive groups which can be used to covalently immobilize biomacromolecules. Two natural macromolecules, chitosan and gelatin, were tethered on these nanofibrous membranes to fabricate dual-layer biomimetic supports for enzyme immobilization in the presence of 1-ethyl-3-(dimethyl-aminopropyl) carbodiimide hydrochloride (EDC)/N-hydroxyl succinimide (NHS). Lipase from Candida rugosa was then immobilized on these dual-layer biomimetic supports using glutaraldehyde (GA), and on the nascent PANCMA fibrous membrane using EDC/NHS as coupling agent, respectively. The properties of the immobilized lipases were assayed. It was found that there is an increase of the activity retention of the immobilized lipase on the chitosan-modified nanofibrous membrane (45.6+/-1.8%) and on the gelatin-modified one (49.7+/-1.8%), compared to that on the nascent one (37.6+/-1.8%). The kinetic parameters of the free and immobilized lipases, K(m) and V(max), were also assayed. In comparison with the immobilized lipase on the nascent nanofibrous membrane, there is an increase of the V(max) value for the immobilized lipases on the chitosan- and gelatin-modified nanofibrous membranes. Results also indicate that the pH and thermal stabilities of lipases increase upon immobilization. The residual activities of the immobilized lipases are 55% on the chitosan-modified nanofibrous membrane and 60% on the gelatin-modified one, after 10 uses.